1. KM71H pHe7 LAHH3,K1 3P 7104815001 25g VLP prep II Bradford assay
Stability Study #1
KM71H pHe7 LAHH3,K1 3P g VLP prep II
Bradford assay
Direct OD measurements
Gels/Blots
ELISA
HPLC

2. Time course for VLP2 stability study
8th Jan
11th Jan
14th Jan
15th Jan
22nd Jan
5th Feb
4th Mar
1st Apr
24th Jun
Day 3 6 7 14 28 56 88
168
Hrs
1hr
4hrs 1d
F/T X
370C RT
40C
-200C
-800C
F/T= freeze/thaw samples. Stored at -20oC and thawed at room temperature x1, x3 or x5 times. Samples are stored and analysed on day 7.
370C samples are stored at -20oC until the assay is carried out. At which point, they are incubated at 37oC for up to 24hrs. Should material be unaffected, this will be extended.
Freeze until day 6 and analyse day 6 & 7.

3. VLP Stability test – general procedure for processing of all samples.
Visual inspection for presence of viable precipitation
2. Centrifugation in microfuge at 13,000 rpm for 10min at 4oC.
3. Any pellet, visible or not, to be re-suspended by vortexing in 600µl buffer (20mM Tris, 5mM EDTA pH 8.4).
4. Supernatant then to be divided for the following assays:
Total protein by Bradford assay
Direct OD measurement at 280nm, 260nm and 320nm. VLP ELISA
SEC-HPLC assay
SDS-Page/Western blotting.
ELISA
10E11 Anti-core to detect the N-terminus
14E11 Anti-core to detect the C-terminus
14C2 Anti-M2 to detect the M2e insert -Developed fro VLP1- Used as negative control in this study
AXLAH In-house anti-LAHH3

4. SDS-PAGE & Western blot
Anti-core Blot
Anti-M2 Blot
Anti-LAHH3 Blot
10E11 ( N-term)
14E11 (C-Term)
14C2
AXLAH
VLP sample is loaded neat or 1:4 diluted
Two gels are required (one for Coomassie and one for WB)
4x2 lanes are used for blotting (ie one neat and one diluted)
The blot is cut into four pieces and developed with the appropriate antibody

5. Antigenicity ELISA
Until SPR becomes available, a relative ELISA will be used.
Three different detection antibodies will be titrated for each sample (anti-core, anti-M2 and anti-LAH).
Anti M2e developed fro VLP1- Used as negative control in this study
The control will be frozen recombinant monomeric core. This is known to be relatively stable.
Results can be expressed relative to the HBc OD.
10E11 Anti-core to detect the N-terminus
14E11 Anti-core to detect the C-terminus
14C2 Anti-M2 to detect the M2e insert-
AXLAH In-house anti-LAHH3
Optimisation
The concentration of primary will be optimised.
Once complete, the VLP concentration will also be optimised.
Optimisation will be carried out using K1:K1 which only expresses core antigen.
Anti-mouse HRP conjugate

6. Liquid Chromatography – Size Exclusion Chromatography (HPLC – SEC)
Instrument: Agilent 1100 HPLC
Column: Agilent Bio-5, 1000A, 4.6x300mm column.
47 nm reference particles Surf-Cal Particle size standard
Run Buffer: 25 mM Tris, pH 7.5, 150 mM NaCl, 1mM CaCl.
Flow rate: 0.30 ml/min. (constant throughout the method)
Column temperature: 30◦C
Injection volume: 10 – 50 µl.
Method duration: 40 minutes.
The column is equilibrated with 0.35 ml/min for 10 minutes before the first sample is injected.
Calibration:
47nm mono-dispersed plastic nanospheres are injected directly onto the column and the A280 and the UV absorbance trace monitored. A single peak is eluted late into the profile. The exact location of the peak is dependent on system configuration (i.e. the length of tubing between the end of the column and the detector). The location of this peak is used as a reference for the location for monodispersed dual core VLP particles. 
Samples:
10 – 50 l of sample is injected and the UV profile monitored. Mono-dispersed VLP will elute with a retention time similar to the reference standard. Large structures/ aggregates will elute earlier.

7. HPLC SEC column
Calibration standards are used to determine VLP size class
Samples are run down the SEC column and then peaks integrated.
Left-most peaks are aggregates
VLP
Aggregate

8. KM71H pHe7 LAHH3,K1 3P 7104815001 25g VLP prep II Time zero
Anti-core Blot
Anti-M2 Blot
AXLAHH3
LAHH3,K1 1:4
Marker
LAHH3,K1
10E11
14E11
14C2
1 sec exposure
1 sec exposure
1 sec exposure
1 sec exposure
Protein concentration by Bradford assay
0.35mg/ml
Direct OD measurements
280nm
260nm
320nm
Neat sample
1.67
1.68
0.53
1:4 dilution
0.57
0.2
1 min exposure
1 min exposure
1 min exposure
1 min exposure

9. . -20C storage has the worse effect on product.
Results summary
Pellet
Total Protein loss
Degradation WB
ELISA
VLP (HPLC) T0 -
+++ ++ + 37
~50% in 6 Days
Freeze/Thaw
~50% in 7 Days
Room Temperature
~30% in 14 Days
+ (14 days)
+4C
~30% in 28 Days
-20C
80-90% in 28 Days
-/+
-80C
~50% in 28 days
. -20C storage has the worse effect on product.
. Freeze/Thaw loss minimised when from -80C.
. Storage buffer needs to be designed to limit the total protein loss during freeze/thaw cycle.

10. 5x 37C-1h RT-Day 14 4C-Day 28 -20C-Day 88 -80C-Day 88 10E11 14E11
LAHH3,K1 1:4
Marker
LAHH3,K1
37C-1h 5x
RT-Day 14
Marker
LAHH3,K1
Marker
LAHH3,K1
LAHH3,K1 1:4
10E11
14E11
AXLAHH3
10E11
14E11
AXLAHH3
10E11
14E11
AXLAHH3
Protein concentration by Bradford assay
0.17 mg/ml
Protein concentration by Bradford assay
0.113 mg/ml
Protein concentration by Bradford assay
0.23mg/ml
4C-Day 28
-20C-Day 88
-80C-Day 88
Marker
LAHH3,K1
LAHH3,K1 1:4
Marker
LAHH3,K1
LAHH3,K1 1:4
Marker
LAHH3,K1
LAHH3,K1 1:4
10E11
14E11
AXLAHH3
10E11
14E11
AXLAHH3
10E11
14E11
AXLAHH3
Protein concentration by Bradford assay
0.235mg/ml
Protein concentration by Bradford assay
0.037mg/ml
Protein concentration by Bradford assay
0.142mg/ml